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Molecular Studies of Hemocyanin Expression in the Dungeness

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Molecular Studies of Hemocyanin Expression in the Dungeness MOLECULAR STUDIES OF HEMOCYANIN EXPRESSION IN THE DUNGENESS CRAB by GREGOR DURSTEWITZ A DISSERTATION Presented to the Department of Biology and the Graduate School of the University of Oregon in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 1996 II "Molecular Studies of Hemocyanin Expression in the Dungeness Crab," a dissertation prepared by Gregor Durstewitz in partial fulfillment of the requirements for the Doctor of Philosophy degree in the Department of Biology. This dissertation has been approved and accepted by: Dr. Nora B. Terwilliger, Chair of the Examining Committee Date committee in charge: Dr. Nora B. Terwilliger, Chair Dr. Roderick capaldi Dr. Ry Meeks-Wagner Eric Schabtach Dr. Kensal van Holde Dr. Tom Stevens Accepted by: Vice Provost and Dean of the Graduate School j i I 1 J iii c 1996 Gregor Durstewitz iv An Abstract of the Dissertation of Gregor Durstewitz for the degree of Doctor of Philosophy in the Department of Biology to be taken June 1996 Title: MOLECULAR STUDIES OF HEMOCYANIN EXPRESSION IN THE DUNGENESS CRAB Approved: Dr. Nora B. Terwilliger This study investigates developmentally regulated changes in the expression of the copper based respiratory protein hemocyanin (Hc) in the Dungeness crab (Cancer magister). Hc gene expression was studied by Northern blot analysis. All six protein subunits were purified and their amino-terminal sequences determined. SUbunit-specific oligonucleotide primers were designed based on these amino­ terminal sequences and on a conserved region near the active site. These primers were then used to PCR-amplify stretches of cDNA coding for developmentally regulated Hc subunit 6 to be used as sUbunit-specific probes in Northern blots. Animals were raised under controlled conditions, and total RNA was isolated from 13 developmental stages and 6 tissue types, run on formaldehyde agarose gels, blotted onto nylon membranes and probed with radioactive 32P-labeled adult Hc- v specific cDNA probes. Results indicate that adult Hc biosynthesis occurs in hepatopancreastissue only and is initiated during the 6lli juvenile instar stage, as indicated by the appearance of subunit 6 mRNA. A model is proposed to explain the observed changes in subunit stoichiometries between juvenile and adult Hc. cDNA coding for developmentally regulated Hc subunit 6 and another putative Hc subunit obtained from a cDNA library screen were sequenced with the dideoxy method. The complete cDNA sequence of subunit 6 showed an open reading frame of 650 amino acids homologous in sequence to other arthropodan Hcs. Functional domains within the proteins were identified, and both were aligned with proteins displaying apparent sequence similarities. A comparison of structural parameters (predicted hydrophilicities, surface probabilities and regional backbone flexibilities) provided evidence for a remarkable degree of structural conservation among crustacean Hcs, chelicerate Hcs, insect hexamerins and arthropodan prophenoloxidases. Parsimony analysis of the aligned sequences allowed a phylogenetic reconstruction of their evolutionary history. Confidence limits were established with the bootstrap approach. The most parsimonious phylogenetic tree consistent with the dataset identified crustacean Hcs, insect hexamerins, chelicerate Hcs and prophenoloxidases as a monophyletic group relative to molluscan Hcs and non- vi arthropodan tyrosinases. Results for individual clades were evaluated and discussed in the light of the evolutionary history of the Hc gene family. , ~ i, j i ~ vii CURRICULUM VITAE NAME OF AUTHOR: Gregor Durstewitz PLACE OF BIRTH: Frankfurt am Main, Germany DATE OF BIRTH: March 30, 1960 GRADUATE AND UNDERGRADUATE SCHOOLS ATTENDED: University of Oregon Freie Universitat Berlin Universitat Tlibingen DEGREES AWARDED: Doctor of Philosophy in Biology, 1996, University of Oregon Diplom (Master of Science) in Biochemistry, 1987, Freie Universitat Berlin Vordiplom (Bachelor of science) in Biochemistry, 1983, Universitat Tlibingen AREAS OF SPECIAL INTEREST: Zoology Marine Biology Natural History PROFESSIONAL EXPERIENCE: Graduate Teaching Fellow, Oregon Institute of Marine Biology and Department of Biology, University of Oregon, Eugene, 1989-1996 Research Assistant, Fritz-Haber-Institut der Max­ Planck-Gesellschaft, Berlin, Germany, 1986-1987 Teaching Assistant, University of Tlibingen Medical School, Tlibingen, Germany, 1985 Military Service, 1978-1980 viii PUBLICATIONS: Brink, L. and Durstewitz, G. (1996) Field guide to marine birds and mammals of Oregon's Bay Area. Oregon Marine Studies Association, Charleston. Durstewitz, G. and Terwilliger, N.B. (1995) Developmental changes in hemocyanin expression in the Dungeness crab: Northern blots and cDNA sequence of a developmentally regulated subunit. Am. Zool. 35, 65A. Durstewitz, G. and Terwilliger, N.B. (1995) Northern blot analysis of the differential expression of hemocyanin subunits in various tissues and developmental stages of the Dungeness crab (Cancer magister). Physiol. Zool. 68, 82. Durstewitz, G., Joslyn, A., Otoshi, C. and Torchin, M. (1993) Guide to intertidal invertebrates. An introduction to tidepool life on the Oregon coast. Oregon Marine Studies Association, Charleston. Durstewitz, G., O'Brien, K. and Terwilliger, N.B. (1992) Specific DNA probes for the analysis of hemocyanin subunit expression in Cancer magister. Am. Zool. 32, 34A. Durstewitz, G. and Tesche, B. (1987) Adsorption of macromolecules onto support films for electron microscopy. Eur. J. Cell BioI. 44, supple 19, 15. Terwilliger, N.B. and Durstewitz, G. (1996) Molecular studies of the sequential expression of a respiratory l protein during crustacean development, in: Molecular Zoology: Advances, strategies and Protocols, eds: J Ferraris, J.D. and Palumbi, S.R., Wiley-Liss, 353-368. j I I f I I ! ix ACKNOWLEDGEMENTS This dissertation is dedicated to my parents Hildegard and Dr. Josef Durstewitz who--sometimes to my surprise-­ have whole-heartedly supported my every move. Many thanks go to my boss Dr. Nora Terwilliger for continuous support throughout the project and for her tolerance in view of my annual northward migrations. I salute my teachers Dr. Christian Bardele and Dr. Wilhelm Harder for inspiring an interest in Zoology and Marine Biology. I am deeply grateful to Dr. Yi-Lin Yan for taking such an interest in my study; without her support and on the spot technical advice in all aspects of molecular biology this study would not have been possible. Bob "Harley" Hanner pointed out the pitfalls of phylogenetic reconstruction. Kristin O'Brien and Dr. Margaret Ryan chipped in with animal husbandry and lab chores. Finally, and maybe most importantly, I thank Clete Otoshi, Kraig Slack and Nicole Apelian for putting up with me and being such good friends through all these years. This study was supported by National Science Foundation grants DCB 8908362 and IBN 9217530 to NBT. x TABLE OF CONTENTS Chapter Page I. INTRODUCTION. ................•............... 1 II. DEVELOPMENT OF PRIMERS AND PROBES FOR ANALYSIS OF CANCER MAGISTER HEMOCYANIN EXPRESSION..... 18 Abstract. .................................. 19 Introduction. .............................. 20 Where is Cancer magister hemocyanin synthesized ?.......................... 28 When does synthesis of adult hemocyanin begin ?................................ 41 Sequencing hemocyanin subunits............. 43 Conclusions. ............................... 44 III. NORTHERN BLOT ANALYSIS OF HEMOCYANIN EXPRESSION IN THE DUNGENESS CRAB - DEVELOPMENTAL CHANGES AND TISSUE-SPECIFIC DIFFERENCES.............. 50 Abstract. .................................. 51 Introduction. .............................. 52 Materials and Methods...................... 55 Results. ................................... 58 Discussion. ................................ 73 IV. cDNA SEQUENCE OF A DEVELOPMENTALLY REGULATED HEMOCYANIN SUBUNIT IN CANCER MAGISTER: IMPLICATIONS FOR THE PHYLOGENY OF THE HEMOCYANIN GENE FAMILy .......•............... 82 Abstract. .................................. 83 Introduction. .............................. 84 Materials and Methods...................... 86 Results 89 II Discussion. ................................ 126 f I V. CONCLUDING SUMMARy........................... 139 I I APPENDIX... .. .. .. .. 142 A. REVERSE TRANSCRIPTION CRT) AND PCR AMPLIFICATION OF HEMOCYANIN mRNA •............ 143 xi B. eDNA CLONING OF PCR PRODUCTS 145 C. NORTHERN BLOTS USING TOTAL RNA FROM DIFFERENT TISSUES AND DEVELOPMENTAL STAGES OF CANCER MAGISTER. ••.............•.•....•.........•..• 146 D. CREATING NESTED DNaseI DELETIONS IN A HEMOCYANIN eDNA.............................. 148 BIBLIOGRAPHY. ........•..........•.......•..•........... 151 xii LIST OF FIGURES Figure Page Chapter I 1. Structure of Limulus Hemocyanin According to X-Ray crystallography at 2.18 A Resolution..•.... 7 2. Electron Micrograph of 25S (Top) and 16S (Bottom) Hemocyanin from the Dungeness Crab..•............ 9 3. Adult Dungeness Crab (Cancer magister) with JuveniIe Instars................................. 12 Chapter II 1. Gel Scan Comparing Five Subunits of Megalopa Hemocyanin (Broad Tracing) and six Subunits of Adult Hemocyanin (Thin Tracing) from Cancer magister Separated by Electrophoresis on 7.5% SOS PAGE......................................... 23 2. Schematic PCR Amplification of Cancer magister eDNA Using Two Different Combinations of Primers. ....................................... .. 31 3. PCR Products on 1.2% Agarose TAE Minigel 34 4. Amino Acid Sequence
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